Preparation And Characterization Of A Novel Polymer Drug Conjugate Based On Poly(?-Malic Acid)

Cancer is a worldwide health problem and chemotherapy is one of the most important means to combat cancer.The currently used chemotherapeutic drugs have many defects including fast metabolism,short half-life,poor water solubility,low bioavailability,and transient high plasma concentration.Polymer drug conjugates are used to load drugs on nano-scale macromolecule carriers and achieve the purpose of killing tumor cells through processes such as blood circulation,tumor tissue enrichment and permeation(EPR effect),cellular uptake and intracellular drug release.Its larger size has also been demonstrated to help overcome multidrug resistance.PMLA is a natural aliphatic polyester which can eventually be degraded to carbon dioxide and water in the tricarboxylic acid cycle in vivo.Furthermore,PMLA could provide many free carboxyl groups that can be covalently linked to a variety of biologically functional groups(eg.multiple drugs,fluorescence tracers,targeting molecules)to form high molecular weight polymer drug conjugates.Hence,PMLA is an efficient carrier of anti-cancer drugs.Single-drug therapy is susceptible to drug resistance,so a synergistic combination therapy has been widely used in clinic.Furthermore,the latest research shows that the application of nanocarriers can increase the efficiency of drug combination therapy and reduce the IC50 value of drugs.Doxorubicin and docetaxel are classic clinical anti-tumor drugs.Clinically,it has been proved that the effect of the two drugs combined is better than that of a single drug.To further improve the efficiency of combined use and overcome the deficiencies of chemotherapeutic drugs,poly(malic acid)is used as a carrier.One end of the chain is linked to m PEG and two chemotherapeutic(doxorubicin and docetaxel)drugs with synergistic anti-tumor effects are connected simultaneously.By controlling the proportion of the drug and the carrier,a polymer drug conjugate with high capacity of drug,long systemic circulation,a stable structure and targetability is achieved.poly(?-malic acid)(PMLA)was synthesized with simple anionic ring-opening polymerization method from our own laboratory.And DOX and DTX were simultaneously conjugated to m PEG-PMLA via a “one pot” method.A polymer drug conjugate,m PEG-PMLA-DOX/DTX,which delivers both drugs at the same time,was constructed and evaluated in vitro.Details are as follows:1.According to the established and optimized route from our laboratory,poly benzyl maloactonate(PMLABn)was obtained through a series of reactions.PMLABn was reacted with m PEG-NH2 to give m PEG-PMLABn.After hydrogenation,m PEG-PMLA was obtained.The doxorubicin and docetaxel were linked via an amide bond and an ester bond respectively.The infrared spectrum of the product was determined by an infrared spectrophotometer and the 1H NMR spectrum demonstrated the successful synthesis of m PEG-PMLA-DOX/DTX.Dox loading was determined by UV-vis,using the molar absorbance at 480 nm.DTX loading was measured by HPLC by totally cleaved from conjugations.When the molar ratio of the two drugs was 1:1(DOX/DTX),the drug loading rates of DOX and DTX were 21.5% and 11.03% respectively for m PEG-PMLA-DOX/DTX conjugate.2.Dynamic dialysis was used to simulate drug release in vitro under p H 7.4 and p H 5.0.Amide bonds was cleaved by cathepsin B to release DOX.After 72 hours,approximately 40% of DOX and 80% of DTX were released.The micelles were prepared from m PEG-PMLA-DOX/DTX by dynamic dialysis.Transmission electron microscopy(TEM)and scanning electron microscopy(SEM)observations showed that the morphology of nanoparticles were uniform and well-dispersed,and the results were consistent with the DLS results.The particle sizes were all around 150 nm.With the increase of drugs conjugation,the zeta potential decreases gradually from-44 m V to-5 m V.The BCA Protein Assay Kit measures the non-specific protein adsorption of carriers and the hemolysis assay further evaluates the biocompatibility of the material.The protein adsorption rate and hemolysis rate were lower than 5% and 2% respectively,indicating that the material has good biocompatibility and can be used as a drug carrier.3.DOX and DTX are considered as first-line therapy for patients with advanced breast cancer in clinic.MDA-MB-231 cells were used to study the synergistic effects.Keep the the total drug concentration of two drugs unchanged,the two drugs are selected in different ratios(20:1,10:1,5:1,2:1,1:1,1:2,1:7)to study anti-tumor effect in vitro.When the molar ratio of DOX to DTX was 10:1,the anti-tumor effect in vitro was better.The cytotoxicity and cell uptake of free drug and polymer drug conjugates were further studied when the molar ratio of the two drugs was 10:1.Dual drug-loaded conjugates could be easier to be internalized by the cells than free drugs.PMLA delivered the chemotherapeutic drugs DOX and DTX simultaneously with high drug loading,which could tolerate the side effects with the efficient anti-tumor effect to make it a good candidate of nanocarrier.And the above results lay the foundation for the next experiments in animal and provide experimental basis for long-circulation,tumor targeting and synergistic effect in vivo of m PEG-PMLA-DOX/DTX.